ABSTRACT The aim of this proposal is to investigate the mechanisms underlying transformation of the mitochondrial voltage-dependent anion channel (VDAC1) from protective to lethal states. We postulate that site-specific phosphorylation of VDAC1 is a key determinant of this transformation. In particular, we will test the hypothesis that phosphorylation of specific sites differentially regulates the gating properties of the channel, and also its ability to associate with pro- and anti-apoptotic proteins. We propose a novel paradigm that this phosphorylation-dependent differential modulation of VDAC1 can shift the balance between cell death and survival. Our studies will establish a mechanistic link between site-specific phosphorylation of VDAC1 and the impact on mitochondrial and cellular functions. In preliminary experiments, we have obtained novel proteomics results that identified candidate sites that impact VDAC1 gating properties and those that regulate its ability to bind anti- and pro-apoptotic proteins. Based on these results, we will systematically target residues that 1) regulate VDAC1 binding with pro- and anti-apoptotic proteins, and 2) alter the channel?s gating characteristics. We will employ a highly orchestrated experimental-computational approach. In Aim 1, we will identify and determine the impact of site-specific phosphorylation on the functional and binding properties of VDAC1. The multifaceted experimental approach will include electrophysiology at the single channel level, mitochondrial bioenergetic measurements, and biochemical and molecular biology approaches. In Aim 2, we will determine the structural impact of site-specific phosphorylation of VDAC1. Molecular dynamics (MD) simulations will be utilized to simulate VDAC1 to characterize the structural changes triggered by site-specific phosphorylation. Free energy calculations of substrate permeation will determine the molecular mechanism that underlies changes in gating due to phosphorylation of specific sites. Comparative MD simulations will be also used to investigate how phosphorylation at more peripheral sites alters the structure, the electrostatics properties affecting membrane/lipid interaction, and dynamics of the putative binding region for hexokinase, a major anti- apoptotic protein that targets VDAC1. In Aim 3, we will test the cardioprotective capacity of VDAC1 mutants in ex vivo and in vivo models. Using ex vivo hearts and in vivo animal models, we will test the transgenic expression of mutant VDAC1 that harbors a specific phosphorylation-resistant site that regulates hexokinase binding or a specific phosphorylated site(s) (phosphomimetic) that promotes cell survival. The proposed research, ranging from the single channel to the in vivo model, will fill a significant void in our understanding of how functional and structural changes in VDAC1 tip the balance between cell survival and cell death. Our long term goal is to delineate how post-translational modifications (PTMs) of VDAC1 contribute to the pathogenesis of ischemic heart disease. These studies could also provide a unique platform for investigating the role of VDAC1 PTMs in other mitochondrial-related diseases, such as Huntington?s or Alzheimer?s diseases.